DESCRIPTION: Endogenous oxalate synthesis plays an important role in idiopathic calcium oxalate stone disease and primary hyperoxaluria. The pathways that underlie this synthesis are poorly understood despite decades of research. In the last funding cycle we established that amino acid and sugar metabolism, previously believed to make major contributions, produce only a limited amount of oxalate. In this application, we propose investigating whether glyoxal is a significant source of oxalate synthesis. Our preliminary studies have shown that glyoxal is converted to oxalate. Glyoxal is primarily converted to glycolate by its interaction with glutathione and the glyoxalase system. Activity of these pathways is contingent on glutathione and NADPH. Any oxidant stress reducing these components could potentially accelerate oxalate synthesis. Three specific aims have been developed. Specific Aim 1: The enzyme(s) potentially catalyzing the conversion of glyoxal to oxalate will be assessed using purified recombinant enzymes and siRNA knockdown in cultured hepatoma (HepG2) cells. The inter-relationship with glyoxal generation and the glyoxalase system will be examined using glutathione depletion. Glucose and linoleate will be assessed as potential sources of glyoxal and oxalate generation using human red cells and HepG2 cells. The use of 13C-isotopes and the quantification of 13C-labelled oxalate, glycolate and glyoxal with ion chromatography coupled to mass detection (IC/MS) or liquid chromatography coupled to mass detection (LC/MS), will allow the flux of carbon to be tracked. Specific Aim 2 will assess if modifying the glyoxalase system impacts oxalate synthesis. It is hypothesized that reducing this system will increase oxalate synthesis. Human erythrocytes and those from Glyoxalase-1 (GLO-1) deficient and wild type mice will be exposed to glyoxal, glutathione depletion and oxidative stress. It is anticipated that these maneuvers will increase oxalate synthesis especially in GLO-1 deficient cells. HepG2 cells, GLO-1 deficient and wild type mice will also be subjected to oxidative stress, glutathione depletion and antioxidants to assess the impact of the glyoxalase system. In Specific Aim 3 the hypothesis that type 2 diabetes is associated with increased endogenous oxalate synthesis will be assessed. Normal adults, those with calcium oxalate stones with and without type 2 diabetes, and diabetics without a history of kidney stones will be characterized. The contribution to endogenous oxalate synthesis will be estimated by placing the participants on tightly controlled low oxalate diets. The relationships between urinary oxalate and glycolate excretion to oxidative stress and glyoxal production will be determined. The Zucker rat, an animal with insulin resistance and recently identified increased oxalate excretion, will be utilized to determine if antioxidant therapy can decrease glyoxal production and urinary glycolate and oxalate. The proposed experiments should produce novel insights into how oxalate is synthesized and should reveal whether antioxidant therapy is a potential therapy to prevent calcium oxalate stone formation.